3D-printing enabled micro-assembly of a microfluidic electroporation system for 3D tissue engineering

Area of Science:

• Biotechnology and Bioengineering
• Tissue Engineering
• Cellular Engineering

Background:

• Electro-transfection is crucial for cellular regulation and tissue engineering but current methods lack in vivo relevance due to limitations in mimicking 3D microenvironments.
• Existing in vitro electro-transfection techniques struggle with controlling 3D electric fields and mass transport in biological matrices, hindering their application in 3D cell cultures.

Purpose of the Study:

• To develop a novel 3D micro-assembly strategy for electro-transfection that overcomes the limitations of conventional methods.
• To create a 3D cell culture system that mimics the in vivo tissue microenvironment for enhanced electro-transfection efficiency.
• To investigate the key parameters influencing transfection efficiency in a 3D culture setting.

Main Methods:

• A 3D printing-assisted micro-assembly strategy was employed to create modular PDMS (polydimethylsiloxane) LEGO® bricks for a 3D cell culture chamber.
• The chamber integrated a 3D perfusion microchannel network for efficient nutrient and waste exchange, facilitating 3D cell growth.
• A multi-directional electric frequency scanning system (3D μ-electro-transfection) was utilized with four electrodes for enhanced cell membrane poration and mass transport.

Main Results:

• The 3D μ-electro-transfection system achieved approximately 15% transfection efficiency with 85% cell viability in 3D cultured HeLa cells, a threefold improvement over conventional methods.
• Electric field strength and plasmid concentration were identified as critical parameters, more influential than pulse duration and duty cycles.
• Successful genetic editing of 3D-cultured Hek-293 cells was demonstrated using CRISPR/Cas9 plasmid delivery, confirming the system's versatility.

Conclusions:

• The 3D-printing enabled micro-assembly strategy provides a facile and effective platform for 3D electro-transfection, mimicking in vivo conditions.
• This novel system significantly enhances gene delivery efficiency and cell viability in 3D cell cultures.
• The developed system holds great potential for versatile gene delivery, cellular engineering, and the development of in vivo-like tissue models for fundamental biological studies.